High-Throughput VDJ Sequencing Is Superior to Quantitative PCR and Flow Cytometry for the Quantification of Minimal Residual Disease In Chronic Lymphocytic Leukemia After Hematopoietic Cell Transplantation.

The primary cause of poor outcome following allogeneic hematopoietic cell transplantation (HCT) for chronic lymphocytic leukemia (CLL) is disease recurrence. Detection of increasing minimal residual disease (MRD) following HCT may permit early intervention such as chemotherapy or donor lymphocyte infusion to prevent clinical relapse. Validated methods for MRD assessment include allele-specific oligonucleotide quantitative PCR (ASO Q-PCR) and flow cytometry (FC). ASO Q-PCR affords high-sensitivity, but is laborious, time-intensive, and not widely available due to its dependence on patient-specific primers. FC is widely available, but is expensive and has lower sensitivity. Boyd et al. (Sci Transl Med 2009;1:12ra23) demonstrated the feasibility of high-throughput sequencing (HTS) of the immunoglobulin heavy chain variable region (IgVH) VDJ segment for quantifying CLL MRD using ASO Q-PCR as a reference standard. In the current study, we compare HTS, ASO Q-PCR, and FC for tracking a group of CLL patients at a series of time points following HCT.

We performed HTS using BIOMED-2 (Van Dongen et al., Leukemia 2003;17:2257) framework 1 (FR1) and 2 (FR2) consensus VH primers with a consensus JH primer. PCR products were sequenced on a 454 pyrosequencer. We tested the sensitivity of both primer sets by creating serial dilutions of a FACS-sorted CLL population into a donor leukapheresis product with known B cell composition such that the CLL cells were as low as 10e-5cells. For our comparisons of HTS, ASO Q-PCR, and FC, we tested a total of 29 samples from 6 CLL patients at up to 10 different time points following HCT.

Although all 6 patients were found to have unmutated IgVH using conventional analysis, we discovered single amino acid sequence variants in up to 17% of CLL-related sequences using HTS. We verified that these changes were not the result of technical artifact by detecting the same oligoclonal species at several time points. For our analyses, we thus defined the “CLL clone” as the dominant sequence plus those sequences of the same CDR3 length that differed by up to one amino acid. In a normal donor, HTS with FR1 and FR2 primers respectively amplified 93.7% and 91.7% of expected V-J combinations (R² = 0.92), indicating broad repertoire coverage. When purified CLL cells were serially diluted to 10e-5 in a normal donor population, we detected the CLL clone with both primer sets when we sequenced the IgVH VDJ with 150,000 reads, confirming 10e-5 sensitivity. ASO Q-PCR demonstrated the same level of sensitivity in this dilution series. In order to compare FC to the DNA-based techniques, we transformed FC data to represent it as CLL cells per microgram of total genomic DNA per cell. FC under-represented MRD levels by up to 80% in comparison to HTS and ASO Q-PCR. The PCR-based techniques were in accord when residual disease was low (R² = 0.88). At high disease burdens, the dynamic range of HTS quantification is limited by the number of sequence reads dedicated to those specimens; however, HTS data permits analysis of CLL clones per total VDJ sequences which appears to be useful for clinical interpretation in such scenarios. Finally, HTS revealed VDJ repertoire reconstitution and hypermutation kinetics. In 3 patients who received post-HCT Rituximab (RTX) for GVHD prophylaxis, 6.0 +/− 0.6% of sequences were hypermutated at day +365 versus 17.8 +/− 3.0% in those who did not receive RTX (p = 0.005), confirming expected delays in immune reconstitution following post-HCT RTX therapy.

HTS exhibits 10e-5 sensitivity for CLL MRD assessment, which is equivalent to ASO Q-PCR and superior to FC. Further validation using material from 35 CLL patients previously evaluated by ASO Q-PCR is underway. We anticipate HTS will broaden the availability of MRD assessment by providing high sensitivity using generally applicable consensus primers. HTS also has the benefits of detecting somatic mutations and revealing rich data regarding VDJ repertoire reconstitution. Furthermore, HTS of B and T cell immunoreceptor genes using consensus primer sets has the potential to provide MRD assessment of all lymphoid malignancies following HCT.

No relevant conflicts of interest to declare.